The valuable specialty chemical succinate and its derivatives have extensive industrial applications. Succinic acid is used as a raw material for food, medicine, plastics, cosmetics, and textiles, as well as in plating and waste-gas scrubbing (61). Succinic acid can serve as a feedstock for such plastic precursors as 1,4-butanediol (BDO), tetrahydrofliran, and gamma-butyrolactone. Further, succinic acid and BDO can be used as monomers for polyesters. If the cost of succinate can be reduced, it will become more useful as an intermediary feedstock for producing other bulk chemicals (47). Along with succinic acid, other 4-carbon dicarboxylic acids such as malic acid and fumaric acid also have feedstock potential.
The production of succinate, malate, and fumarate from glucose, xylose, sorbitol, and other “green” renewable feedstocks (in this case through fermentation processes) is an avenue to supplant the more energy intensive methods of deriving such acids from nonrenewable sources. Succinate is an intermediate for anaerobic fermentations by propionate-producing bacteria but those processes result in low yields and concentrations. It has long been known that mixtures of acids are produced from E. coli fermentation. However, for each mole of glucose fermented, only 1.2 moles of formic acid, 0.1-0.2 moles of lactic acid, and 0.3-0.4 moles of succinic acid are produced. As such, efforts to produce carboxylic acids fermentatively have resulted in relatively large amounts of growth substrates, such as glucose, not being converted to desired product.
Numerous attempts have been made to metabolically engineer the anaerobic central metabolic pathway of E. coli to increase succinate yield and productivity (7, 8, 12, 14, 15, 20, 24, 32, 44, 48). Genetic engineering coupled with optimization of production conditions have also been shown to increase succinate production. An example is the growth of a succinate producing mutant E. coli strain using dual phase fermentation production mode which comprises an initial aerobic growth phase followed by an anaerobic production phase or/and by changing the headspace conditions of the anaerobic fermentation using carbon dioxide, hydrogen or a mixture of both gases (35, 49).
Specifically, manipulating enzyme levels through the amplification, addition, or reduction of a particular pathway can result in high yields of a desired product. Various genetic improvements for succinic acid production under anaerobic conditions have been described that utilize the mixed-acid fermentation pathways of E. coli. One example is the overexpression of phosphoenolpyruvate carboxylase (pepc) from E. coli (34). In another example, the conversion of fumarate to succinate was improved by overexpressing native fumarate reductase (frd) in E. coli (17, 53). Certain enzymes are not indigenous in E. coli, but can potentially help increase succinate production. By introducing pyruvate carboxylase (pyc) from Rhizobium etli into E. coli, succinate production was enhanced (14, 15, 16). Other metabolic engineering strategies include inactivating competing pathways of succinate. When malic enzyme was overexpressed in a host with inactivated pyruvate formate lyase (pfl) and lactate dehydrogenase (ldh) genes, succinate became the major fermentation product (44, 20). An inactive glucose phosphotransferase system (ptsG) in the same mutant strain (pfl- and ldh-) had also been shown to yield higher succinate production in E. coli and improve growth (8).
The maximum theoretical yield (molar basis) of succinate from glucose under anaerobic conditions is limited to 1 mol/mol, assuming that all the carbon flux will go through the native succinate fermentative pathway (FIG. 1). The fermentative pathway converts oxaloacetate (OAA) to malate, fumarate and then succinate and this pathway requires 2 moles of NADH per mole of succinate produced. One major obstacle to high succinate yield through the fermentative pathway is due to NADH limitation. This is because one mole of glucose can provide only two moles of NADH through the glycolytic pathway; however, the formation of one mole of succinate through the native fermentative pathway requires two moles of NADH. Anaerobic production of succinate is also hampered by the limitations of slow cell growth and production.
Metabolic engineering has the potential to considerably improve process productivity by manipulating the throughput of metabolic pathways. Specifically, manipulating enzyme levels through the amplification, addition, or deletion of a particular pathway can result in high yields of a desired product. What is needed in the art is an improved bacterial strain that produces higher levels of succinate and other carboxylic acids than heretofor provided.